Biologics manufacturing mostly relies on host organisms, such as Chinese hamster ovary (CHO) cells and Escherichia coli, to produce therapeutic proteins. While these host cells produce these target therapeutic molecules, they also continue to produce other biological molecules necessary for their survival, metabolism and other functions, commonly referred to as host cell proteins (HCPs). Unlike process-related contaminants that can often be minimized through process optimization, the presence of HCPs is an inherent part of biologics production and must be carefully monitored and controlled.
HCPs represent more than just background noise in the manufacturing process; they can have real consequences. If their levels are not controlled, HCPs can potentially impact product quality and even lead to adverse patient reactions.
The presence of HCPs in drugs can have an immunogenic effect and lead to adverse immune responses in patients, lead to reduced product efficacy due to the degradation of excipients, decrease the product shelf life, or affect efficacy by altering the glycan profile. Lastly, HCPs may have proteolytic activity and degrade therapeutic proteins, further undermining the drug’s effectiveness.
To mitigate these risks, HCP presence is closely monitored and controlled during the manufacturing process for clinical and commercial supply, as required by regulatory authorities.[1, 2] However, HCP clearance strategy should be planned for products used in preclinical studies for toxicological assessments and the early process development stage during cell line construction. By detecting and addressing HCP-related issues early, developers can streamline process development, avoid costly setbacks later on, and ultimately improve patient safety.
As someone who not only works in this industry but is also a potential patient, I find this especially important. Every drug on the shelf isn’t just a product; it’s a potential lifeline. I expect therapies to be safe, effective, and high-quality. That’s why I believe that CDMOs (Contract Development and Manufacturing Organizations) have a critical responsibility. Their commitment to quality through thorough impurity monitoring, process development, high-quality standards, and strict adherence to regulatory regulations directly translates into safer treatments for everyone.
Monitoring HCPs
HCPs comprise thousands of different proteins present at very low concentrations, making detection and quantification challenging. Depending on the project-specific needs and requirements, our team at Lonza uses a multitude of analytical methods for quantifying HCP levels in drug products:
- Sandwich ELISA with polyclonal HCP antibodies remains the gold standard immunoassay, recommended by regulatory authorities for HCP analysis due to its high sensitivity and robustness.[5] With good-quality HCP antibodies and HCP standards, sandwich ELISA is the most popular and preferred heterogeneous format for HCP ELISA. HCP ELISA is developed and validated for several assay parameters, including accuracy, precision, limit of quantitation, and robustness.
- HCP immunoassay development (Sandwich ELISA) HCP immunoassays depend highly on the quality of HCP antibodies used, which should recognize HCPs as broadly as possible. Therefore, HCP antibody development is the core of a good HCP immunoassay, and its main focus is to define the ELISA suitability recommended by the regulatory authorities.[3,4]
- Immunocoverage is a critical attribute of HCP antibodies and measures how many HCPs in the total population in the manufacturing process can be recognized. The same HCP antibody preparation can show different coverage data with different evaluation methods, including some using native conditions. Two-dimensional (2D) Western blotting is a main workhorse for HCP immunocoverage assessment[2] but often gives a lower result compared to other methods.
- While ELISA is highly effective, it doesn’t identify individual HCPs or detect those present in very low amounts. For that, mass spectrometry (MS) is increasingly used as a complementary method. MS can precisely identify and quantify specific HCPs, including ones that may pose a higher risk. It’s especially valuable during process development and risk assessment. Recent improvements—like the use of stable isotope-labelled peptides[6] and standardized reference materials—have made MS more sensitive and consistent. However, it remains a complex and costly technique, and is not yet practical for routine quality control testing.
By combining these different techniques, manufacturers can gain a more complete understanding of HCP presence and behavior, helping to ensure biologic therapies are both safe and reliable for patients. As the industry evolves, many future customers will bring their own cell lines into established manufacturing platforms through “drop-in” approaches. To support this model, we are developing a holistic HCP service offering that can adapt to a wide range of processes and ensure robust impurity control from early development through to commercial production. In parallel, we are actively working on new mass spectrometry–based methods to address the industry-wide challenge of accurately identifying and quantifying problematic HCPs, pushing beyond current limitations to support safer, high-quality biologics.
References
- [1] USP "1132", Residual host cell protein Measurement in biopharmaceuticals. May 2016
- [2] EP general chapter 2.6.34, Host-cell protein assays. April 2017.
- [3] Baldus, et al., Comparison of purification strategies for antibodies used in a broad spectrum host cell protein immunoassay. Biotechnology and Bioengineering. 2018;115: 413–422.
- [4] G. Mao. Compositions and methods for the detection of host cell proteins. US patent, US11353468B2. June 2022.
- [5] Wang et al, Host-Cell Protein Measurement and Control. BioPharm International, 2015; 28 (6):32–38
- [6] S Liberatori, Analysis of host-cell proteins in in-process samples by next-generation proteomics combined with targeted absolute quantitation. BEPBA HCP conference, May 2024, Maryland.